The lack of methods to image functioning retinal circuitry in the living eye is a fundamental impediment to all efforts at vision restoration. The ability to assess the structure and function of retinal circuitry in vivo at multiple retinal layer simultaneously will not only transform our understanding of where different vision restoration strategies succeed and fail, the feedback these tools will provide will greatly shorten the development cycle for all approaches in which they are deployed. Investigators in the Advanced Retinal Imaging Alliance (ARIA) at the University of Rochester (Jennifer Hunter, Bill Merigan, and David Williams) will create a new retinal imaging tool designed to track changes in structure and function in both the inner and outer retina at cellular resolution in individual animals over time. This tool will combine two-photon, adaptive optics imaging of genetically-encoded calcium indicators to track the neuronal responses of individual photoreceptors and ganglion cells in two different animal models, mouse and monkey. We will assess the value of this imaging technology in three different approaches to vision restoration. Applying these tools to a gene therapy approach, the Rochester team will collaborate with Connie Cepko's laboratory at Harvard University to rescue cone function in retinitis pigmentosa through the up-regulation of anti-oxidants. We will demonstrate the value of these imaging tools for an optogenetics approach by expressing channel-rhodopsin in monkey cones in collaboration with Botond Roska at the Miescher Institute for Biomedical Research. This effort will also develop a primate model of retinal degeneration in which a viral vector is used to shave off photoreceptor outer segments. Finally, these imaging tools will be deployed to monitor the differentiation and neural connectivity of stem cells in mouse and then monkey in collaboration with David Gamm at the University of Wisconsin, Madison. This consortium will not only develop advanced imaging technology, it will translate it to the field of vision restoration, and create a collaborative environment for sharing best practices and combining different approaches.